Antimalarial potential of Moringa oleifera Lam. (Moringaceae): A review of the ethnomedicinal, pharmacological, toxicological, and phytochemical evidence

Several regions of the world frequently use the species Moringa oleifera Lam. (Moringaceae) in traditional medicine. This situation is even more common in African countries. Many literature reports point to the antimalarial potential of this species, indicating the efficacy of its chemical compounds against malaria-causing parasites of the genus Plasmodium. From this perspective, the present study reviews the ethnobotanical, pharmacological, toxicological, and phytochemical (flavonoids) evidence of M. oleifera, focusing on the treatment of malaria. Scientific articles were retrieved from Google Scholar, PubMed®, ScienceDirect®, and SciELO databases. Only articles published between 2002 and 2022 were selected. After applying the inclusion and exclusion criteria, this review used a total of 72 articles. These documents mention a large use of M. oleifera for the treatment of malaria in African and Asian countries. The leaves (63%) of this plant are the main parts used in the preparation of herbal medicines. The in vivo antimalarial activity of M. oleifera was confirmed through several studies using polar and nonpolar extracts, fractions obtained from the extracts, infusion, pellets, and oils obtained from this plant and tested in rodents infected by the following parasites of the genus Plasmodium: P. berghei, P. falciparum, P. yoelii, and P. chabaudi. Extracts obtained from M. oleifera showed no toxicity in preclinical tests. A total of 46 flavonoids were identified in the leaves and seeds of M. oleifera by different chromatography and mass spectrometry methods. Despite the scarcity of research on the antimalarial potential of compounds isolated from M. oleifera, the positive effects against malaria-causing parasites in previous studies are likely to correlate with the flavonoids that occur in this species.(AU)

Biofilm model on mice skin wounds

Purpose: To evaluate a biofilm model of Pseudomonas aeruginosa in excisional cutaneous wound in mice. Methods: Preclinical, translational study conducted with 64 C57BL/6 mice randomly assigned to control and intervention groups. Evaluation was on days D0, D3, D5, D7 and D10 of wound making. The profile of biofilm formation and induction was evaluated using wound closure kinetics, quantitative culture, and evaluation of wounds using transmission electron microscopy (TEM). Clinical evaluation was performed by liver tissue culture, weight variation, and quantification of leukocytes in peripheral blood. Analyses were performed with GraphPad Prism software. Results: Bacterial load for induction of infection with P. aeruginosa and survival of animals was 104 UFC·mL-1. In D5 (p < 0.0001) and D7 (p < 0.01), animals in the intervention group showed a delay in the healing process and had their wounds covered by necrotic tissue until D10. Statistical differences were observed in wound cultures and weight at D5 and D7 (p < 0.01). Liver cultures and leukocyte quantification showed no statistical differences. No bacteria in planktonic or biofilm form were identified by TEM. Conclusions: The findings raise questions about the understanding of the ease of formation and high occurrence of biofilm in chronic wounds.

The use of occlusive dressings: influence on excisional wound healing in animal model

Purpose: To analyze the influence of occlusive dressing on the healing of excisional skin wounds in mice. Methods: Pre-clinical, comparative, and translational study. Mice were divided into three experimental groups: wounds occluded with hydrocolloid (HD) dressings, transparent polyurethane film (TF) dressings, and without occlusion (WO), monitored at three, six and 14 days, with eight animals each. Closure rate, infiltration of neutrophils and macrophages, measurement of tumor necrosis factor-α (TNF-α) and vascular endothelial growth factor (VEGF) and, histologically, angiogenesis were evaluated. Results: Wound closure was accelerated in the occlusive groups. There was a decrease in TNF-α levels in the HD group when compared to the WO and TF groups. Neutrophils accumulation decreased in the HD group. Increased dosages of macrophages were evidenced in the HD group, compared to the WO and TF groups. Levels of VEGF were increased in the TF and HD groups. Conclusions: It is suggested that the occlusion of wounds modulates the inflammatory response.

Secretion pattern of canine amniotic stem cells derived extracellular vesicles

Extracellular vesicles (EVs) derived from stem cells (SCs) have regenerative potential and the possibility of being used in treating chronic diseases. EVs present lower risk of tumorigenicity and easily to isolation and storage. Therefore, this research aims to compare the morphological characteristics of the EVs (up to 150nm) derived from stem cells obtained from canine amniotic membranes in different passages during the in vitro culture. For this, cells from the amniotic membranes were isolated, cultured, and characterized. In order to answer our aim, the number of cells was normalized at each passage to generate conditioned media for EVs separation. The cells were differentiated into adipogenic, chondrogenic, and osteogenic tissue, to characterize these cells as mesenchymal stem cells (MSC). Moreover, flow cytometry analysis was performed and showed that the MSC were positive for CD90, CD105 and negative for CD34, CD45, mesenchymal and hematopoietic markers, respectively. For EVs analysis, MSC in different passages (P0-P2) were culture until 80% of confluence, then the medium was replaced by EVs depleted medium. After 48h, culture medium was collected and centrifuged to separate EVs, followed by nanoparticle tracking analysis. The EVs were also characterized by western blot and transmission electron microscopy (TEM). EVs were positive for Alix and negative for Cytochrome C as well as presented the traditional cup-shape by transmission electronic microscopy. Our results demonstrated that the concentration in the different passages was increased in P0 compared to P1 and P2 (p<0.05). No differences were found in EVs size (P0=132nm, P1=130nm and P2=120nm). Together, these results demonstrate that P0 of MSC is enriched of EVs when compared to later passages, suggesting that this passage would be the best to be applied in pre-clinical tests. Despite that, more studies are necessary to identify the EVs content and how the cells will respond to treatment with them.(AU)

King Cobra and snakebite envenomation: on the natural history, human-snake relationship and medical importance of Ophiophagus hannah

Abstract King Cobra (Ophiophagus hannah) has a significant place in many cultures, and is a medically important venomous snake in the world. Envenomation by this snake is highly lethal, manifested mainly by neurotoxicity and local tissue damage. King Cobra may be part of a larger species complex, and is widely distributed across Southeast Asia, southern China, northern and eastern regions as well as the Western Ghats of India, indicating potential geographical variation in venom composition. There is, however, only one species-specific King Cobra antivenom available worldwide that is produced in Thailand, using venom from the snake of Thai origin. Issues relating to the management of King Cobra envenomation (e.g., variation in the composition and toxicity of the venom, limited availability and efficacy of antivenom), and challenges faced in the research of venom (in particular proteomics), are rarely addressed. This article reviews the natural history and sociocultural importance of King Cobra, cases of snakebite envenomation caused by this species, current practice of management (preclinical and clinical), and major toxinological studies of the venom with a focus on venom proteomics, toxicity and neutralization. Unfortunately, epidemiological data of King Cobra bite is scarce, and venom proteomes reported in various studies revealed marked discrepancies in details. Challenges, such as inconsistency in snake venom sampling, varying methodology of proteomic analysis, lack of mechanistic and antivenomic studies, and controversy surrounding antivenom use in treating King Cobra envenomation are herein discussed. Future directions are proposed, including the effort to establish a standard, comprehensive Pan-Asian proteomic database of King Cobra venom, from which the venom variation can be determined. Research should be undertaken to characterize the toxin antigenicity, and to develop an antivenom with improved efficacy and wider geographical utility. The endeavors are aligned with the WHO´s roadmap that aims to reduce the disease burden of snakebite by 50% before 2030.

King Cobra and snakebite envenomation: on the natural history, human-snake relationship and medical importance of Ophiophagus hannah

King Cobra (Ophiophagus hannah) has a significant place in many cultures, and is a medically important venomous snake in the world. Envenomation by this snake is highly lethal, manifested mainly by neurotoxicity and local tissue damage. King Cobra may be part of a larger species complex, and is widely distributed across Southeast Asia, southern China, northern and eastern regions as well as the Western Ghats of India, indicating potential geographical variation in venom composition. There is, however, only one species-specific King Cobra antivenom available worldwide that is produced in Thailand, using venom from the snake of Thai origin. Issues relating to the management of King Cobra envenomation (e.g., variation in the composition and toxicity of the venom, limited availability and efficacy of antivenom), and challenges faced in the research of venom (in particular proteomics), are rarely addressed. This article reviews the natural history and sociocultural importance of King Cobra, cases of snakebite envenomation caused by this species, current practice of management (preclinical and clinical), and major toxinological studies of the venom with a focus on venom proteomics, toxicity and neutralization. Unfortunately, epidemiological data of King Cobra bite is scarce, and venom proteomes reported in various studies revealed marked discrepancies in details. Challenges, such as inconsistency in snake venom sampling, varying methodology of proteomic analysis, lack of mechanistic and antivenomic studies, and controversy surrounding antivenom use in treating King Cobra envenomation are herein discussed. Future directions are proposed, including the effort to establish a standard, comprehensive Pan-Asian proteomic database of King Cobra venom, from which the venom variation can be determined. Research should be undertaken to characterize the toxin antigenicity, and to develop an antivenom with improved efficacy and wider geographical utility. The endeavors are aligned with the WHO´s roadmap that aims to reduce the disease burden of snakebite by 50% before 2030.(AU)

Anatomical description of the skulls of peccaries (Tayassu tajacu, Linnaeus 1758) by computed tomography / [Descrição anatômica de crânio de catetos (Tayassu tajacu, Linnaeus, 1758) por tomografia computadorizada]

The aim of this study was to evaluate the anatomical structures of the skulls of peccaries to establish the basis for their clinical study and future preclinical research. Ten skulls of adult peccaries were subjected to tomographic examination. The data obtained were processed via three-dimensional image reconstruction software (3D images). The reconstructions obtained from the neurocranium of the studied specimens allowed the identification and description of the following structures: nasal bone, frontal bone, parietal bones, incisor bone, maxillary bone, zygomatic bone, temporal bone, palatal bone, occipital bone, vomer bone, pterygoid bone, sphenoid bone, paranasal sinuses and orbit. Computed tomography proved to be an important diagnostic tool in the investigation of the skull of this species, allowing the acquisition of anatomical values not yet documented for the species in the literature.(AU)

O objetivo deste estudo foi avaliar as estruturas anatômicas dos crânios de catetos, a fim de se estabelecerem as bases para seu estudo clínico e futuras pesquisas pré-clínicas. Dez crânios de catetos adultos foram submetidos a exame tomográfico. Os dados obtidos foram introduzidos em um software de reconstrução de imagens tridimensionais (imagens em 3D). As reconstruções obtidas do neurocrânio dos espécimes estudados permitiram a identificação e a descrição das seguintes estruturas: osso nasal, osso frontal, ossos parietais, osso incisivo, osso maxilar, osso zigomático, osso temporal, osso palatino, osso occipital, osso vômer, osso pterigoide, osso esfenoide, seios paranasais e órbita. A tomografia computadorizada mostrou-se como uma ferramenta diagnóstica importante na investigação do crânio dessa espécie, permitindo a aquisição de valores anatômicos ainda não documentados para a espécie na literatura.(AU)

Biomodelos e a covid-19: estado da arte e tendências de uso / Biomodels and covid-19: state-of-the-art and usage trends

A COVID-19 é uma nova coronovirose que demonstrou seu potencial pandêmico a partir de um surto observado em dezembro de 2019 na cidade de Wuhan, na China e, atualmente, já está sendo compreendida como uma síndrome de disfunção múltipla de órgãos, além dos pulmões. Até o momento, há ainda muito a se conhecer sobre o vírus, suas interações e potencial de transmissão, bem como a patogênese da doença; o desenvolvimento das candidatas a vacinas, terapias, diagnósticos mais precisos e ferramentas de controle da doença. Um dos principais desafios encontrados atualmente na pesquisa em COVID-19 é a disponibilização de modelos animais que mimetize a fisiopatogênese da doença humana. Sendo assim, este artigo objetiva fazer uma revisão não só do estado da arte em estudos pré-clínicos desenvolvidos para entendimento e enfrentamento da COVID-19 com a participação de diversos modelos animais de experimentação no aprimoramento de terapias imunes, mas também trazer à discussão a participação de espécies animais selvagens na origem da pandemia, ciclo de transmissão e manutenção da doença. De acordo com a revisão dos trabalhos publicados, foi possível observar que algumas espécies, como primatas não humanos e camundongos têm potencial de uso em múltiplas formas de experimentação, enquanto outras se limitam a um tipo de observação. Desta forma, a avaliação contínua de bons modelos animais para estudo da COVID-19 consolida a importância da investigação em animais e dos estudos pré-clínicos para solucionar esta e próximas pandemias que possam vir a ocorrer na história da saúde pública mundial.

COVID-19 is a new virus disease that demonstrated its pandemic potential from an outbreak observed in December 2019, in Wuhan, China and it is now considered as a multiple organ dysfunction syndrome, in addition to the lungs. So far, there is still much to know about the virus, its interactions and potential for transmission, as well as the pathogenesis of the disease; the development of vaccines candidates, therapies, more accurate diagnoses and disease control tools. One of the main challenges currently found in research in COVID-19 is the availability of animal models that mimic the pathophysiology of human disease. Therefore, this article aims to review not only the state of the art in preclinical studies developed to understand and confront COVID-19 with participation of several animal models of experimentation in the improvement of immune therapies, but also to bring to discussion the participation of wild animal species in the origin of the pandemic, transmission cycle and maintenance of the disease. According to this review, it was possible to observe that some species, such as non-human primates and mice, have potential for use in multiple forms of experimentation, while others are limited to one type of observation. Thus, the continuous evaluation of good animal models for COVID-19 studies consolidates the importance of animal research and preclinical studies to solve this and the next pandemics that may occur in the history of public health worldwide.

Biomodelos e a covid-19: estado da arte e tendências de uso / Biomodels and covid-19: state-of-the-art and usage trends

A COVID-19 é uma nova coronovirose que demonstrou seu potencial pandêmico a partir de um surto observado em dezembro de 2019 na cidade de Wuhan, na China e, atualmente, já está sendo compreendida como uma síndrome de disfunção múltipla de órgãos, além dos pulmões. Até o momento, há ainda muito a se conhecer sobre o vírus, suas interações e potencial de transmissão, bem como a patogênese da doença; o desenvolvimento das candidatas a vacinas, terapias, diagnósticos mais precisos e ferramentas de controle da doença. Um dos principais desafios encontrados atualmente na pesquisa em COVID-19 é a disponibilização de modelos animais que mimetize a fisiopatogênese da doença humana. Sendo assim, este artigo objetiva fazer uma revisão não só do estado da arte em estudos pré-clínicos desenvolvidos para entendimento e enfrentamento da COVID-19 com a participação de diversos modelos animais de experimentação no aprimoramento de terapias imunes, mas também trazer à discussão a participação de espécies animais selvagens na origem da pandemia, ciclo de transmissão e manutenção da doença. De acordo com a revisão dos trabalhos publicados, foi possível observar que algumas espécies, como primatas não humanos e camundongos têm potencial de uso em múltiplas formas de experimentação, enquanto outras se limitam a um tipo de observação. Desta forma, a avaliação contínua de bons modelos animais para estudo da COVID-19 consolida a importância da investigação em animais e dos estudos pré-clínicos para solucionar esta e próximas pandemias que possam vir a ocorrer na história da saúde pública mundial.(AU)

COVID-19 is a new virus disease that demonstrated its pandemic potential from an outbreak observed in December 2019, in Wuhan, China and it is now considered as a multiple organ dysfunction syndrome, in addition to the lungs. So far, there is still much to know about the virus, its interactions and potential for transmission, as well as the pathogenesis of the disease; the development of vaccines candidates, therapies, more accurate diagnoses and disease control tools. One of the main challenges currently found in research in COVID-19 is the availability of animal models that mimic the pathophysiology of human disease. Therefore, this article aims to review not only the state of the art in preclinical studies developed to understand and confront COVID-19 with participation of several animal models of experimentation in the improvement of immune therapies, but also to bring to discussion the participation of wild animal species in the origin of the pandemic, transmission cycle and maintenance of the disease. According to this review, it was possible to observe that some species, such as non-human primates and mice, have potential for use in multiple forms of experimentation, while others are limited to one type of observation. Thus, the continuous evaluation of good animal models for COVID-19 studies consolidates the importance of animal research and preclinical studies to solve this and the next pandemics that may occur in the history of public health worldwide.(AU)

Image-guided study of swine anatomy as a tool for urologic surgery research and training

Purpose: To describe the anatomy of the swine urinary system using computed tomography and to discuss the role of this animal as an experimental model for urological procedures. Methods: Three male Landrace pigs underwent computed tomography and the anatomy of the urinary system and renal circulation was analyzed and described. Results: In all animals, 2 kidneys, 2 ureters and one bladder were identified. Each kidney presented a single renal artery vascularization, with a mean diameter on the right of 4.45 and 5.31 mm on the left (p < 0.0001) and single renal vein drainage, with a mean diameter on the right of 5.78 and 5.82 mm on the left (p = 0.0336). The average renal length was 9.85 cm on the right and 10.30 cm on the left (p < 0.0001). The average renal volume was 113.70 cm3 on the right and 109.70 cm3 on the left (p 0.0001). The average length of the ureter was 19.78 cm on the right and 22.08 cm on the left (p < 0.0001). The average bladder volume was 423.70 cm3. Conclusions: The data obtained show similarities with human anatomy, suggesting the viability of the swine model for planning preclinical trials, basic research, refinement in experimental surgery and surgical training for urological procedures.(AU)

Dalbergia ecastaphyllum leaf extracts: in vitro inhibitory potential against enzymes related to metabolic syndrome, inflammation and neurodegenerative diseases

For the first time, the anti-hemolytic activity and the enzyme inhibitory activities of Dalbergia ecastaphyllum leaves extracts were tested against α-amylase, α-glucosidase, lipase, acetylcholinesterase, butyrylcholinesterase, tyrosinase and hyaluronidase. The phenolic profile of the obtained extracts was also investigated by high-performance liquid chromatography with photodiode array detection (HPLC-PAD). The extracts showed inhibitory activity against all enzymes evaluated, with the highest inhibitory activity reported for the enzyme hyaluronidase (28.28 ± 2.43 to 72.19 ± 1.40 μg mL-1). The obtained extracts also demonstrate anti-hemolytic activity (52.22 ± 1.62 to 71.17 ± 1.82%). Among the phenolic compounds identified, protocatechuic, vanillic and β-resorcylic acids were the most abundant (1.13 ± 0.06 to 2.53 ± 0.06, 0.90 ± 0.06 to 2.19 ± 0.06 and 1.03 ± 1.62 to 22.11 ± 1.62 mg L-1, respectively). In the statistical analysis, a significant correlation was found between the flavonoids content and all enzymes inhibitory activities. The present study showed that D. ecastaphyllum leaves extracts may have the potential to be used in the therapeutic treatment of several diseases such as Alzheimer, Parkinson, type 2 diabetes mellitus, hyperglycemia, and pigmentation, as well as those associated with oxidative stress.

Multiple uses of fibrin sealant for nervous system treatment following injury and disease

Lesions to the nervous system often produce hemorrhage and tissue loss that are difficult, if not impossible, to repair. Therefore, scar formation, inflammation and cavitation take place, expanding the lesion epicenter. This significantly worsens the patient conditions and impairment, increasing neuronal loss and glial reaction, which in turn further decreases the chances of a positive outcome. The possibility of using hemostatic substances that also function as a scaffold, such as the fibrin sealant, reduces surgical time and improve postoperative recovery. To date, several studies have demonstrated that human blood derived fibrin sealant produces positive effects in different interventions, becoming an efficient alternative to suturing. To provide an alternative to homologous fibrin sealants, the Center for the Study of Venoms and Venomous Animals (CEVAP, Brazil) has proposed a new bioproduct composed of certified animal components, including a thrombin-like enzyme obtained from snake venom and bubaline fibrinogen. Thus, the present review brings up to date literature assessment on the use of fibrin sealant for nervous system repair and positions the new heterologous bioproduct from CEVAP as an alternative to the commercial counterparts. In this way, clinical and pre-clinical data are discussed in different topics, ranging from central nervous system to peripheral nervous system applications, specifying positive results as well as future enhancements that are necessary for improving the use of fibrin sealant therapy.(AU)

ESTUDOS DE TOXICOLOGIA PRÉ CLÍNICA DE UMA FORMULAÇÃO CONTENDO DERIVADOS DE CANNABIS SATIVA

O Brasil apresenta historicamente em seu cotidiano o uso de plantas e substâncias com ações calmantes, analgésicas dentre outros efeitos. Avanços na pesquisa com plantas medicinais apresentaram efetividade de tais substâncias, e o uso de ativos extraídos de plantas como a Cannabis sativa, maconha como é conhecida popularmente, tem demonstrado eficácia em tratamentos diversos, sendo uma alternativa em potencial para doenças do sistema nervoso central, porém seu uso é limitado devido à questões políticas e culturais. O objetivo do presente estudo foi realizar a comprovação de segurança de uma formulação contendo Canabidiol (CBD) 200mg/ml em solução de administração oral, com testes de avaliação pré-clínica que visam constatar possíveis alterações de parâmetros bioquímicos, hematológicos, alterações em órgãos alvo de metabolismo e avaliação do perfil reprodutivo dos animais a serem testados. Ratos e coelhos foram submetidos à tratamentos agudos e crônicos com diferentes dosagens da formulação de CBD, calculadas via extrapolação alométrica (33, 99 e 297 mg/Kg via oral, e 297 mg/Kg intraperitoneal em ratos, e 17, 51 e 153 mg/Kg via oral, e 153 mg/Kg intraperitoneal em coelhos). Todos protocolos experimentais foram realizados de acordo com as diretrizes da Organization for Economic Co-Operation and Development OECD e Agência Nacional de Vigilância Sanitária ANVISA, bem como também com aprovação no Comitê de Ética em Pesquisa Envolvendo Animais CEPEA da UNIPAR. Os números de animais utilizados em ambos os protocolos foram minimizados de acordo com as diretrizes internacionais em relação à redução de animais utilizados em pesquisa. Testes de toxicidade aguda e crônica aplicados em ambas espécies utilizadas neste estudo demostraram segurança da formulação nas doses e vias administradas, pois não ocorreram alterações significativas nos parâmetros avaliados, bem como não foram observadas macro e microscopicamente alterações nos órgãos alvos de metabolismo. Após realização dos protocolos de toxicidade reprodutiva, não se observou alteração nos parâmetros de fertilidade e reprodução, bem como também nos fetos oriundos das fêmeas submetidas aos tratamentos propostos. Tais resultados nos fornecem preditivos para a segurança da formulação com CBD 200 mg/ml para administração oral, de acordo com os protocolos experimentais realizados neste estudo. Os testes pré-clínicos fazem parte de uma etapa dentro da grande complexidade que é o processo para obtenção do registro de medicamento a partir de substâncias ativas. Tais testes são primordiais, pois são eles que nos indicam a possibilidade da continuidade dos estudos clínicos com substâncias ativas.

Brazil has historically presented in its daily life the use of plants and substances with calming actions, analgesics, among other effects. Advances in research with medicinal plants have shown the effectiveness of such substances, and the use of actives extracted from plants such as Cannabis sativa, marijuana as it is popularly known, has shown efficacy in several treatments, being a potential alternative for diseases of the central nervous system, however its use is limited due to political and cultural issues. The aim of this study was to prove the safety of a formulation containing Cannabidiol (CBD) 200mg/ml in a solution for oral administration, with pre-clinical evaluation tests aimed at verifying possible changes in biochemical and hematological parameters, changes in target organs of metabolism and evaluation of the reproductive profile of the animals to be tested. Rats and rabbits were subjected to acute and chronic treatments with different dosages of the CBD formulation, calculated via allometric extrapolation (33, 99 and 297 mg/kg orally, and 297 mg/kg intraperitoneally in rats, and 17, 51 and 153 mg /kg orally, and 153 mg/kg intraperitoneally in rabbits). All experimental protocols were carried out in accordance with the guidelines of the Organization for Economic Co- Operation and Development OECD and the National Health Surveillance Agency ANVISA, as well as with approval by the Ethics Committee on Research Involving Animals CEPEA at UNIPAR. The numbers of animals used in both protocols were minimized in accordance with international guidelines regarding the reduction of animals used in research. Acute and chronic toxicity tests applied to both species used in this study demonstrated the safety of the formulation in the doses and routes administered, as there were no significant changes in the evaluated parameters, as well as macro and microscopic changes in Organs target organs of metabolism were not observed. After carrying out the reproductive toxicity protocols, there was no change in fertility and reproduction parameters, as well as in fetuses from females submitted to the proposed treatments. These results provide us with predictors for the safety of the formulation with CBD 200 mg/ml for oral administration, according to the experimental protocols carried out in this study. Pre-clinical tests are part of a step within the great complexity of the process for obtaining drug registration from active substances. Such tests are essential, as they indicate the possibility of continuing clinical studies with active substances.

AVALIAÇÃO DA TOXICIDADE AGUDA DA BIOMASSA MICELIAL DO Ganoderma lucidum (Curt.: Fr.) Karst. BIOACUMULADA COM LÍTIO EM RATOS

O transtorno bipolar, um distúrbio mental crônico caracterizado por episódios maníacos e/ou depressivos, afeta mais de 60 milhões de pessoas em todo o mundo. O tratamento é crônico e sintomático e tem como principal fármaco representante o carbonato de lítio. Porém, devido ao estreito índice terapêutico, este medicamento pode provocar muitos efeitos adversos, como hipotiroidismo, tremor muscular involuntário, polidipsia, poliúria, diarreia, dispneia, ganho de peso e rash cutâneo. Assim, o controle da absorção e biodisponibilidade do lítio é fundamental para reduzir a dose do fármaco e consequentemente atenuar seus efeitos adversos. Uma alternativa promissora para o controle destes fatores é a acumulação do medicamento em macromoléculas presentes na biomassa micelial de cogumelos que poderiam retardar a absorção do lítio ao longo do sistema digestório. Um destes cogumelos é o Ganoderma lucidum (Curt.: Fr.) Karst. (Basidiomycota), um dos cogumelos medicinais mais populares do mundo, que tem amplo potencial de bioacumular metais de interesse nutricional e farmacológico em seu micélio. Entretanto, previamente aos estudos pré-clínicos para comprovação da eficácia desta substância, estudos de toxicidade devem ser conduzidos. Desta forma, esta pesquisa teve como objetivo avaliar a intensidade da toxicidade ocasionada pela administração aguda da biomassa micelial de G. lucidum bioacumulada com lítio em ratos Wistar, de acordo com o protocolo 423 da Organização para a Cooperação e Desenvolvimento Econômico (OECD). Todo o protocolo experimental foi aprovado pelo Comitê de Ética Envolvendo Pesquisa e Experimentação Animal da UNIPAR (protocolo nº 1006/2019). A biomassa micelial de G. lucidum foi produzida em frascos Erlenmeyer contendo meio líquido de cultivo à base de melaço de cana-de-açúcar e solução de carbonato de lítio, mantidos em cultivo por 21 dias, em estufa. Para o estudo de toxicidade, dois grupos de ratos Wistar machos de 2 meses (n = 6), foram tratados com dose única de 300 mg/kg ou 2000 mg/kg de biomassa micelial de G. lucidum bioacumulada com lítio, por gavagem. Ratos Wistar machos tratados agudamente com veículo (água deionizada) compuseram o grupo basal (n = 6). A avaliação dos sinais clínicos e peso corporal dos animais foi realizada 30 minutos, 1, 2, 3 e 4 horas e 14 dias após o tratamento. No 15º dia, após a eutanásia dos animais por aprofundamento anestésico em câmara de saturação com isoflurano, o sangue foi coletado por decapitação para investigação de parâmetros bioquímicos e hematológicos. Na sequência, rins, fígado, baço e coração foram coletados, pesados e processados para análises histopatológicas. Não foram observadas diferenças significativas no peso dos animais, sinais clínicos e parâmetros bioquímicos, hematológicos e histopatológicos. Conclui-se, assim, que ratos tratados com a biomassa micelial de G. lucidum bioacumulada com lítio não apresentam sinais de toxicidade. Entretanto, estudos de toxicidade com doses repetidas devem ser conduzidos para a determinação do perfil de toxicidade desta substância.

Bipolar disorder, a chronic mental disorder characterized by manic and/or depressive episodes, affects more than 60 million people worldwide. The treatment is chronic and symptomatic, and its main representative drug is lithium carbonate. However, due to the narrow therapeutic index, this medication can cause many adverse effects, such as hypothyroidism, involuntary muscle tremor, polydipsia, polyuria, diarrhea, dyspnoea, weight gain and skin rash. Thus, controlling the absorption and bioavailability of lithium is essential to reduce the dose of the drug and consequently mitigate its adverse effects. A promising alternative for the control of these factors, is the accumulation of the drug in macromolecules present in the mycelial biomass of mushrooms that could delay the absorption of lithium throughout the digestive system. One of these mushrooms is Ganoderma lucidum (Curt.: Fr.) Karst. (Basidiomycota), one of the most popular medicinal mushrooms in the world, which has ample potential to accumulate metals of nutritional and pharmacological interest in its mycelium. However, prior to preclinical studies to prove the efficacy of this substance, toxicity studies must be conducted. Thus, this research evaluated the intensity of toxicity caused by the acute administration of the mycelial biomass of G. lucidum accumulated with lithium in Wistar rats, according to protocol 423 of the Organization for Economic Cooperation and Development (OECD). The entire experimental protocol was approved by the Ethics Committee Involving Animal Research and Experimentation at UNIPAR (protocol nº 1006/2019). The mycelial biomass of G. lucidum was produced in Erlenmeyer flasks containing liquid culture medium based on sugarcane molasses and lithium carbonate solution, kept in culture for 21 days, in an oven. For the toxicity study, two groups of 2-month-old male Wistar rats (n = 6) were treated with a single dose of 300 mg/kg or 2000 mg/kg of mycelial biomass of G. lucidum accumulated with lithium, by gavage. Male Wistar rats treated acutely with vehicle (deionized water) composed the basal group (n = 6). The evaluation of clinical signs and body weight of the animals was performed 30 minutes, 1, 2, 3 and 4 hours and 14 days after treatment. On the 15th day, after euthanizing the animals by deepening anesthetic in a saturation chamber with isoflurane, blood was collected by decapitation to investigate biochemical and hematological parameters. Then, kidneys, liver, spleen, and heart were collected, weighed, and processed for histopathological analysis. There were no significant differences in the animals' weight, clinical signs, and biochemical, hematological, and histopathological parameters. It is concluded, therefore, that rats treated with the mycelial biomass of G. lucidum accumulated with lithium do not show signs of toxicity. However, toxicity studies with repeated doses should be conducted to determine the toxicity profile of this substance.

DOENÇA DE PARKINSON: AVALIAÇÃO DA DOR E EFEITOS CLÍNICOS DO TRATAMENTO COM Bacopa monnieri EM PACIENTES

A doença de Parkinson (DP) é uma enfermidade crônica e progressiva do sistema nervoso central decorrente da perda de neurônios dopaminérgicos da substância negra. A DP é caracterizada pelos sinais cardinais de rigidez, bradicinesia, instabilidade postural e tremor. Além dos sintomas motores, quadros álgicos são frequentes e podem ocorrer distúrbios do sono, disfunção cognitiva e depressão, repercutindo em baixa qualidade de vida, tornando a doença ainda mais incapacitante. Neste sentido, tratamentos paliativos são aplicados para aliviar os sintomas da DP e melhorar a qualidade de vida do paciente. Já a abordagem farmacológica, padrão-ouro, é feita com levodopa, uma forma efetiva de restaurar a neurotransmissão, mas que a longo prazo pode ocasionar efeitos adversos. Assim, a busca por novas terapias eficazes e seguras, capazes de atuar sobre as alterações dopaminérgicas, cognitivas e motoras é fundamental. Neste contexto, a Bacopa monnieri, uma planta utilizada para melhora do desempenho cognitivo, é uma alternativa promissora. Tradicionalmente esta planta é utilizada para melhora do desempenho cognitivo. Estudos pré-clínicos apontam prevenção da neurodegeneração em modelo de DP e estudos clínicos evidenciam ações positivas sobre a função cognitiva. Entretanto, ainda não há investigações clínicas sobre os efeitos desta planta em pacientes com DP. Assim, esta pesquisa foi dividida em duas etapas. Na primeira, investigou-se o tempo de evolução, incidência do locus corporal e interferência da dor na qualidade de vida de pacientes com DP. Foi aplicada a Escala Visual Analógica e o Inventário Breve de Dor de Wisconsin em 37 pacientes com DP, com idade média de 68 anos. Detectou-se dor em 87,5% dos participantes, com maior frequência nos membros superiores e interferência no desempenho das atividades gerais. Na segunda etapa, 20 pacientes com DP com idade média de 76 anos foram recrutados para um estudo clínico intervencionista, controlado, paralelo e duplo-cego a fim de investigar os efeitos da B. monnieri em sua função motora e qualidade de vida. Os pacientes foram tratados com comprimidos de B. monnieri (225 ou 450 mg/paciente) ou placebo (comprimidos com as mesmas características físico-químicas, sem a droga vegetal). A qualidade de vida e a função motora foram avaliadas pelo questionário de Qualidade de Vida na doença de Parkinson (PDQL) e pela seção III da Escala Unificada de Avaliação da doença de Parkinson (MDS-UPDRS), respectivamente, antes, 30, 60 e 90 dias após o tratamento. Nenhuma diferença foi observada nos sintomas parkinsonianos, sistêmicos, função emocional ou social. O delta percentual (%) indicou melhorias tempo-dependentes na função emocional decorrentes de tratamentos com o uso da B. monnieri (450 mg). Encontrou-se forte correlação entre qualidade de vida e sintomas motores no início e após 30 dias de tratamento com B. monnieri, e uma correlação moderada para 60 e 90 dias de tratamento com B. monnieri quando comparado com o tempo inicial. Sugere-se que o tratamento com B. monnieri possa melhorar a função emocional de pacientes com DP. Entretanto, ensaios clínicos com mais pacientes são necessários para confirmar este achado. Ainda, destaca-se que o tratamento da DP em todas as dimensões é necessário para aliviar a dor e, consequentemente, melhorar a qualidade de vida dos pacientes.

Parkinson's disease (PD) is a chronic and progressive disease of the central nervous system resulting from the loss of dopaminergic neurons of the substantia nigra. PD is characterized by cardinal signs of stiffness, bradykinesia, postural instability, and tremor. In addition to motor symptoms, pain is common and sleep disorders, cognitive dysfunction and depression may occur, resulting in low quality of life, making the disease even more disabling. In this sense, palliative treatments are applied to relieve the symptoms of PD and improve the patient's quality of life. The gold standard pharmacological approach, on the other hand, is done with levodopa, an effective way to restore neurotransmission, but which in the long run can cause adverse effects. Thus, the search for new effective and safe therapies, capable of acting on dopaminergic, cognitive, and motor changes is fundamental. In this context, Bacopa monnieri is a promising alternative. Traditionally this plant is used to improve cognitive performance. Pre-clinical studies indicate prevention of neurodegeneration in a PD model and clinical studies show positive actions on cognitive function. However, there are still no clinical investigations on the effects of this plant on PD patients. Thus, this research was divided into two stages. In the first, the evolution time, incidence of the body locus and pain interference in the quality of life of PD patients were investigated. The Visual Analogue Scale and the Wisconsin Brief Pain Inventory were applied to 37 PD patients, with a mean age of 68 years. Pain was detected in 87.5% of the participants, most frequently in the upper limbs and interference in the performance of general activities. In the second stage, 20 PD patients, with an average age of 76 years, were recruited for a controlled, parallel, double-blind clinical intervention study to investigate the effects of B. monnieri on their motor function and quality of life. The patients were treated with B. monnieri tablets (225 or 450 mg/patient) or placebo (tablets with the same physicochemical characteristics, without the plant drug). Quality of life and motor function were assessed using the Quality of Life in Parkinson's Disease questionnaire (PDQL) and section III of the Unified Parkinson's Disease Assessment Scale (MDS-UPDRS), respectively, before 30, 60 and 90 days after treatment. No difference was observed in parkinsonian, systemic symptoms, emotional or social function. The percentage delta (%) indicated time-dependent improvements in emotional function resulting from treatment with B. monnieri (450 mg). A strong correlation was found between quality of life and motor symptoms at the beginning and after 30 days of treatment with B. monnieri, and a moderate correlation for 60 and 90 days of treatment with B. monnieri when compared to the initial time. It is suggested that treatment with B. monnieri may improve the emotional function of patients with PD. However, clinical trials with more patients are needed to confirm this finding. Still, it is highlighted that the treatment of PD in all dimensions is necessary to relieve pain and, consequently, improve the quality of life of patients.

INVESTIGAÇÃO ETNOFARMACOLÓGICA DOS EFEITOS HEPATOPROTETORES DE Croton urucurana BAILL. EM ROEDORES

A doença hepática gordurosa associada ao metabolismo (DHGAM), uma condição caracterizada pelo acúmulo de lipídeos no fígado, pode progredir e levar a complicações como cirrose, insuficiência hepática, hepatocarcinoma celular e problemas cardiovasculares. Dentre os fatores de risco para a enfermidade, a obesidade e a dislipidemia são identificadas como fatores de risco independentes, podendo ainda atuar de maneira sinérgica para o desenvolvimento ou severidade da doença quando associadas ao tabagismo ou à hipertensão arterial sistêmica. Embora a DHGAM acometa um quarto da população mundial, não há terapia medicamentosa aprovada para a enfermidade e, mesmo que novos tratamentos estejam em estudo, as taxas de resposta terapêutica são modestas. De encontro com esta necessidade de desenvolvimento de fármacos, é inegável que as plantas medicinais são importantes fontes para a descoberta de novos componentes bioativos para a formulação de medicamentos. Neste contexto, destaca-se a Croton urucurana, árvore cujas folhas, cascas do tronco e seiva são amplamente utilizadas popularmente para tratar doenças cardiovasculares, úlceras gástricas, diarreia e distúrbios hepáticos. No entanto, a eficácia desta espécie medicinal contra doenças hepáticas ainda não foi investigada farmacologicamente. Assim, esta pesquisa investigou os efeitos da fração solúvel em etanol das folhas da C. urucurana em um modelo animal de DHGAM associada à hipertensão, tabagismo e dislipidemia. Para tanto, ratos espontaneamente hipertensos receberam dieta enriquecida com 0,5% de colesterol e foram expostos à fumaça de cigarro (9 cigarros por dia, 5 dias por semana), por 10 semanas. Nas últimas 5 semanas, os animais (n = 8) foram tratados por via oral (gavagem) com veículo (água filtrada; grupo controle negativo), extrato de C. urucurana (30, 100 e 300 mg/kg) e sinvastatina (2,5 mg/kg) + enalapril (15 mg/kg). Um grupo de ratos Wistar normotensos não exposto aos fatores de risco e tratados com veículo também foi avaliado (grupo basal, n = 8). Ao término do tratamento, o sangue foi coletado para análise dos níveis de colesterol, triglicerídeos, alanina aminotransferase (ALT) e aspartato aminotransferase (AST). O fígado foi coletado para análise histopatológica. Os níveis de colesterol e triglicerídeos também foram avaliados no fígado e nas fezes. O modelo de estudo induziu DHGAM, aumentou os níveis plasmáticos de AST e ALT, aumentou os níveis plasmáticos, hepáticos e fecais de colesterol e triglicerídeos, induziu estresse oxidativo e alterações celulares hepáticas no grupo controle negativo. O tratamento com o extrato de C. urucurana (300 mg/kg) e sinvastatina + enalapril diminuiu os níveis plasmáticos e hepáticos de colesterol e triglicerídeos e, em contraste com o tratamento com sinvastatina + enalapril, o tratamento com a dose mais alta de C. urucurana reverteu as alterações celulares hepáticas e reduziu efetivamente os níveis de AST e ALT. Conclui-se que a C. urucurana exerce importantes efeitos hepatoprotetores pré-clínicos e pode ser útil para o tratamento de pacientes com doença hepática gordurosa associada ao metabolismo.

Metabolic associated fatty liver disease (MAFLD), a condition characterized by hepatic accumulation of lipids, can progress and lead to complications such as cirrhosis, liver failure, cellular hepatocarcinoma and cardiovascular problems. Among the risk factors for illness, obesity and dyslipidemia are identified as independent risk factors, and they can also act synergistically for the development or severity of the disease when associated with smoking or systemic arterial hypertension. Although MAFLD affects a quarter of the world population, there is no approved drug therapy for the disease and, even if new treatments are being studied, the rates of therapeutic response are modest. In view of this need for drug development, it is undeniable that medicinal plants are important sources for the discovery of new bioactive components for the formulation of medicines. In this context, Croton urucurana, a tree whose leaves, bark and sap are widely used to treat cardiovascular diseases, gastric ulcers, diarrhea, and liver disorders stands out. However, the effectiveness of this medicinal species against liver disease has not yet been investigated pharmacologically. Thus, this research investigated the effects of the ethanol-soluble fraction of C. urucurana leaves in an animal model of MAFLD associated with hypertension, smoking and dyslipidemia. Therefore, spontaneously hypertensive rats received a diet enriched with 0.5% cholesterol and were exposed to cigarette smoke (9 cigarettes a day, 5 days a week), for 10 weeks. In the last 5 weeks, the animals (n = 8) were treated orally (gavage) with vehicle (negative control group), C. urucurana extract (30, 100 and 300 mg/kg) and simvastatin (2.5 mg/kg) + enalapril (15 mg/kg). A group of normotensive Wistar rats not exposed to risk factors and treated with vehicle was also evaluated (baseline group, n = 8). At the end of treatment, blood was collected for analysis of cholesterol, triglyceride, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. The liver was collected for histopathological analysis. Cholesterol and triglyceride levels were also assessed in the liver and feces. The study model induced MAFLD, increased plasma levels of AST and ALT, increased plasma, hepatic and fecal levels of cholesterol and triglycerides, induced oxidative stress and liver cell changes in the negative control group. Treatment with 300 mg/kg C. urucurana and simvastatin + enalapril decreased plasma and hepatic cholesterol and triglycerides and, in contrast to treatment with simvastatin + enalapril, the highest dose of C. urucurana reversed hepatic cellular changes and effectively reduced levels of liver of AST and ALT. In conclusion, C. urucurana has important pre-clinical hepatoprotective effects and can be useful for the treatment of patients with Metabolic associated fatty liver disease.

Desenvolvimento de vacinas inativadas contra a Língua Azul e a Doença Epizoótica Hemorrágica para cervídeos: ensaios pré-clínicos.

A língua azul e a doença epizoótica hemorrágica são viroses caracterizadas pela ocorrência de distúrbios hemorrágicos em cervídeos e são responsáveis por impactos negativos na conservação dessas espécies. Essas doenças apresentam como agentes etiológicos os vírus BTV e EHDV, respectivamente. A vacinação é o método mais eficaz de controle e prevenção dessas viroses, entretanto, no Brasil, vacinas contra o BTV e o EHDV não são licenciadas pelos órgãos competentes e não há vacinas disponíveis para cervídeos no mercado nacional e exterior. Em razão do exposto, o principal objetivo dessa pesquisa foi o desenvolvimento de vacinas e a realização de ensaios pré-clínicos com formulações vacinais contendo amostras de BTV-3, BTV-14, BTV-18, BTV-19, BTV-22, BTV-24, EHDV-1 e EHDV-2 isoladas na reserva biológica da Itaipu Binacional. As suspensões virais obtidas em laboratório foram inativadas pela beta-propriolactona, adjuvadas de 10% de hidróxido de alumínio e 40% de saponina e distribuídas em duas vacinas distintas: vacina 1 (sorotipos BTV-3, BTV-14, BTV-18 e BTV-19) e vacina 2 (sorotipos BTV-22, BTV-24, EHDV-1 e EHDV-2). Em seguida, testes de inativação viral, esterilidade, inocuidade e potência foram realizados. O teste de inativação viral confirmou a inativação completa dos vírus, enquanto o teste de esterilidade demonstrou a ausência de crescimento bacteriano. O teste de inocuidade, realizado em quatro coelhos, não evidenciou nenhum efeito adverso à vacinação. Para a realização do teste de potência, foram utilizados 15 coelhos distribuídos equitativamente em três grupos experimentais. Os animais foram vacinados isoladamente (G1 Vacina 1; G2 Vacina 2) e associadamente (G3 Vacinas 1+2) com três doses, administradas aos 0, 21 e 51 dias de experimentação. As mensurações de anticorpos totais e anticorpos neutralizantes do teste de potência foram realizadas, respectivamente, por meio de imunodifusão em gel de ágar (IDGA), utilizando as amostras coletadas aos 81 dias de experimentação, e soroneutralização em microplaca, utilizando as amostras coletadas aos 0, 21, 51 e 81 dias de experimentação. Os resultados do IDGA indicaram a presença de anticorpos totais em 100% dos animais testados para BTV, demonstrando que os antígenos foram capazes de induzir resposta imune humoral. Não foram realizados testes de IDGA para o EHDV. Já no teste de soroneutralização, as medianas obtidas indicaram ausência de anticorpos neutralizantes para o EHDV e presença de anticorpos neutralizantes para o vírus BTV-18 (na vacinação isolada), e BTV-18, BTV-19 e BTV-24 (na vacinação associada) nas amostras coletadas no 81º dia de experimentação. Foi possível observar que as formulações desenvolvidas não foram capazes de induzir resposta de anticorpos neutralizantes para todos os sorotipos e que a vacinação associada promoveu melhor resposta imune humoral. Nossos resultados permitirão o desenvolvimento de novas pesquisas para a formulação de vacinas para cervídeos brasileiros.

Bluetongue and epizootic hemorrhagic are viruses diseases characterized by the occurrence of hemorrhagic disorders in deer and are responsible for negative impacts on these species conservation. These diseases have BTV and EHDV viruses, respectively, as their etiologic agents. Vaccination is the most effective method of controlling and preventing these viruses. However, in Brazil, vaccines against BTV and EHDV are not licensed by regulatory organizations and there are no vaccines available for deer in national or foreign markets. In view of the above, the main objective of this research was the development of vaccines and the performance of pre-clinical trials with vaccine formulations containing samples of BTV-3, BTV-14, BTV-18, BTV-19, BTV22, BTV -24, EHDV-1 and EHDV-2 isolated in the biological reserve of Itaipu Binacional. The viral suspensions obtained in the laboratory were inactivated by beta-propiolactone, adjuvanted with 10% aluminum hydroxide and 40% saponin and distributed in two different vaccines: vaccine 1 (serotypes BTV-3, BTV-14, BTV-18 and BTV -19) and vaccine 2 (serotypes BTV-22, BTV-24, EHDV-1 and EHDV-2). Then, viral inactivation, sterility, inoculation, and potency tests were performed. The viral inactivation test confirmed complete virus inactivation, while the sterility test demonstrated the absence of bacterial growth. The inoculation test, carried out on four rabbits, did not show any adverse effects to vaccination. To perform the potency test, 15 rabbits were equally distributed in three experimental groups. The animals were vaccinated separately (G1 - Vaccine 1; G2 - Vaccine 2) and in combination (G3 - Vaccines 1 + 2) with three doses, administered at 0, 21 and 51 days of experimentation. The measurements of total antibodies and neutralizing antibodies of the potency test were carried out, respectively, by means of agar gel immunodiffusion (AGID), using the samples collected after 81 days of experimentation, and soroneutralization in microplate, using the samples collected at 0, 21, 51 and 81 days of experimentation. The results of the AGID indicated the presence of total antibodies in 100% of the animals tested for BTV, demonstrating that the antigens were able to induce humoral immune response. No AGID tests were performed for EHDV. In the soroneutralization test, the medians obtained indicated the absence of neutralizing antibodies for EHDV and the presence of neutralizing antibodies for BTV-18 virus (in the isolated vaccination), and BTV-18, BTV-19 and BTV-24 viruses (in the associated vaccination) in samples collected on the 81st day of experimentation. It was possible to observe that the formulations developed were not able to induce neutralizing antibody response for all serotypes and that the associated vaccination promoted a better humoral immune response. Our results will allow the development of new research for the formulation of vaccines for Brazilian deer

Interação entre células tronco de membrana amniótica e tumor mamário canino in vitro

Tumores nas glândulas mamárias caninas representam a neoplasia mais comum em cadelas. Este tipo de tumor em cadelas é muito semelhante quanto ao comportamento biológico, resposta a agentes citotóxicos e características histológicas comparadas aos tumores apresentados em humanos, mas sua taxa de incidência é três vezes maior que em mulheres. Em relação aos tratamentos, muitas vezes é necessária a utilização da quimioterapia para erradicar metástases, no entanto, a maioria dos tumores caninos é apenas moderadamente sensível à quimioterapia. Nesse contexto, o atual cenário favorece ensaios terapêuticos pré-clinicos e pesquisas voltadas para novos tratamentos anti-câncer. Nesse sentido, o uso de células-tronco amnióticas tem se destacado neste campo. Desta forma, caracterizamos células de membrana amniótica canina, onde verificamos que estas apresentavam expressão para marcadores de células mesenquimais. Além disso, apresentaram capacidade de diferenciação em linhagens osteogênica, adipogênica e condrogênica e, quando injetadas in vivo, não apresentaram potencial tumorigênico. Posteriormente, avaliamos a matriz extracelular de carcinomas mamários canino do tipo tubular e sólido. Verificamos que o carcinoma sólido apresentou maior grau de malignidade devido a suas características histológicas e com os componentes presentes em sua matriz extracelular. Por fim, avaliamos os efeitos do tratamento com doxorrubicina (DOXO) em associação com células-tronco de membrana amniótica canina (AMC) sobre células de carcinoma inflamatório mamário canino (IPC-366), e células de carcinoma sólido mamário canino (TCM). Para isso, quatro grupos experimentais foram analisados: grupo controle sem tratamento; grupo I com DOXO, grupo II com AMC e grupo III associação de DOXO com AMC. Verificamos que nas células IPC-366, o grupo associação de DOXO com AMC em células apresentou melhores resultados em relação à redução do crescimento celular e na expressão de proteínas relacionadas à proliferação e angiogênese. Já nas células TCM verificamos que o tratamento com células AMC, sendo ou não associada à DOXO, possui a capacidade de prender as células no ciclo celular na fase de repouso fazendo com que as mesmas apresentem redução em seu crescimento e proliferação celular.

The tumors on the canine mammary glands represent the most common neoplasm in bitches. This type of tumor in bitches responds to cytotoxic agents in a very similar biological way when compared to tumors presented in humans. Besides that, they have the same histological characteristics, but its incidence rate is three times higher in bitches than in women. Regarding treatments, chemotherapy is often required to eradicate metastases, however, most canine tumors are only moderately sensitive to chemotherapy. In this context, the current scenario favors pre-clinical therapeutic trials and research aimed at new anticancer treatments. In this sense, the use of amniotic stem cells has been outstanding in this field. So, we characterized canine amniotic membrane cells. At this characterization we verified that they had expression for markers of mesenchymal cells, and present differentiation capacity in osteogenic, adipogenic and chondrogenic lines. When injected in vivo, did not present tumorigenic potential. Subsequently, we evaluated the extracellular matrix of canine mammary carcinomas of the tubular and solid type. We verified that solid carcinoma showed a higher degree of malignancy due to its histological characteristics and the components present in its extracellular matrix. Finally, we evaluated the effects of doxorubicin treatment (DOXO) in combination with canine amniotic membrane (AMC) stem cells on canine mammary inflammatory carcinoma cells (IPC-366), and solid canine mammary carcinoma cells. For this, four experimental groups were analyzed: control group without treatment; group I with DOXO, group II with AMC and group III association of DOXO with AMC. We found that at the IPC-366 cells, the association group of DOXO with AMC in cells presented better results regarding to the reduction of cell growth and expression of proteins related to proliferation and angiogenesis. In the TCM cells, we found that treatment with AMC cells, whether associated with DOXO, can bind the cells in the cell cycle during the resting phase, promoting the cell growth decrease and proliferation. Keywords: Amniotic membrane. Canine breast cancer. Doxorubicin. Neoplasm. Stem cells.

AVALIAÇÃO ECOCARDIOGRÁFICA EM MODOS B, M E DOPPLER E PADRONIZAÇÃO DO STRAIN E STRAIN RATE POR SPECKLE TRACKING BIDIMENSIONAL EM CUTIAS (Dasyprocta prymnolopha, WAGLER, 1831) CONTIDAS QUIMICAMENTE COM CETAMINA E XILAZINA

As cutias (Dasyprocta prymnolopha, Wagler, 1831) são roedores silvestre amplamente encontrados em toda a América e têm se destacado como bons modelos animais para investigações científicas. No entanto, assim como na maioria das espécies silvestres, o manuseio destes animais com fins científicos só é possibilitado por meio da contenção química ou até mesmo de anestesia geral. Este trabalho teve por objetivo estudar a função cardiovascular de cutias contidas quimicamente, pela realização de exames ecocardiográficos e mensuração do strain e strain rate por speckle tracking para obtenção de valores de referência para a espécie e verificar a aplicabilidade destes animais como modelos para o estudo de alterações cardiovasculares em seres humanos. Foram utilizados 16 exemplares, contidos pela associação de cloridrato de cetamina e xilazina e submetidas ao exame ecocardiográfico transtorácico. Para obtenção das mensurações padrões, foram realizadas imagens em modos B, M e Doppler. Realizou-se a análise da deformação miocárdica radial e longitudinal do ventrículo esquerdo. A janela paraesternal direita foi utilizada para acesso ao eixo curto cardíaco e a esquerda para acesso ao eixo longo. Seis perfis radiais de valores de strain (ST) e strain rate (SR) foram obtidos, correspondendo à média dos valores para cada segmento. Foram analisados um total de 18 segmentos do miocárdio para cada espécime e a média de valores foi utilizada para compor a análise estatística. A comparação das médias entre os gêneros não demonstrou diferença estatística, sendo os demais tratamentos estatísticos baseados na amostra total de 16 animais. As variáveis que demonstraram coeficiente de correlação estatisticamente significantes em relação ao peso foram: AE, AE/AO, FC, AVmax, onda E, E/TRIV (p>0,05). A FC apresentou correlação positiva com o SIVd (r = 0,51), EPSS (r = 0,55) e onda E (r = 0,68); negativa com o TRIV (r = -0,41), onda A (r = -0.54), AVmax (r = -0,53) e Pmax (r = -0,65). A presente pesquisa permitiu a obtenção dos valores presumidamente normais das dimensões de câmaras e paredes cardíacas, strain e strain rate por speckle tracking bidimensional de cutias contidas quimicamente com cetamina e xilazina. As semelhanças observadas com a espécie humana para os valores de função cardíaca, obtidos pela técnica de strain por speackle tracking em cutias, sugerem que esta espécie apresente potencial para ser explorada como modelo animal em estudos pré-clínicos em cardiologia.

The agouti (Dasyprocta prymnolopha, Wagler, 1831) are wild rodents widely found throughout America, and have stood out as good animal models for scientific investigations. However, as in most wild species, the handling of these animals for scientific purposes is only possible through chemical containment or even general anesthesia. The objective of this study was to study the cardiovascular function of the chemo contained in the samples by performing echocardiographic tests and measuring the strain and strain rate by speckle tracking to obtain reference values for the species and verify the applicability of these animals as models for the study of cardiovascular changes in humans. Sixteen specimens, contained by the combination of ketamine hydrochloride and xylazine, were submitted to transthoracic echocardiography. To obtain the standard measurements, B, M and Doppler mode images were performed. The analysis of the radial and longitudinal myocardial deformation of the left ventricle was carried out. The right parasternal window was used for access to the short cardiac axis and the left for access to the long axis. Six radial profiles of strain (ST) and strain rate (SR) values were obtained, corresponding to the mean of the values for each segment. A total of 18 myocardial segments were analyzed for each specimen and the mean values ere used to compose the statistical analysis. The comparison between the means between the genders did not show statistical difference, being the other statistical treatments based on the total sample of 16 animals. The variables that showed statistically significant correlation coefficients in relation to weight were: LA, LA/AO, HR, AVmax, E 'wave, E/IVRT (p>0.05). HR showed a positive correlation with SIVd (r = 0.51), EPSS (r = 0.55) and E 'wave (r = 0.68); negative with IVRT (r = -0.41), wave A (r = -0.54), AVmax (r = -0.53) and Pmax (r = -0.65). The present research allowed to obtain presumed normal values of the chambers and heart walls, strain and strain rate by two-dimensional speckle tracking of chews containing chemically with ketamine and xylazine. The similarities observed with the human species for cardiac function values obtained by speackle tracking strain in agouti suggest that this species has the potential to be exploited as an animal model in preclinical studies in cardiology.

Avaliação da interação biológica entre compósito de nanotubo de carbono, quitosana e hidroxiapatita e tecido ósseo São Paulo 2019

Atualmente têm se desenvolvido substitutos ósseos, com propósito de auxiliar na regeneração óssea em casos de perda tecidual. Para avaliação da biocompatibilidade e osteocondução, é necessária sua implantação in vivo, em animais de laboratório e grandes animais, para aprovação em uso clínico futuro. Ratos e ovinos são modelos experimentais em engenharia tecidual. Podem ser adicionados aos biomateriais, células-tronco mesenquimais para potencialização do efeito regenerativo ósseo. O objetivo desse estudo foi avaliar a biocompatibilidade e osteocondução de compósito de nanotubo de carbono, quitosana e hidroxiapatita (CNQH), enriquecidos ou não com células-tronco mesenquimais oriundas de medula óssea de ovino (CTMO) em ratos e ovinos. Inicialmente, sessenta ratos foram divididos em grupos, sendo que o implante de CNQH com ou sem CTMO, em subcutâneo, foi realizado em 20 animais (submetidos a eutanásia após 7 e 30 dias da cirurgia), comparando com 10 animais controle, e em 20 animais em defeito na calvária (submetidos a eutanásia após 20 e 60 dias da cirurgia) comparando com 10 animais controle. Foram realizadas avaliações histológicas de tecido subcutâneo e calvária, após eutanásia, comparando-se os grupos. Posteriormente, dezoito ovelhas foram submetidas à ostectomia em tíbia e osteossíntese com placa de compressão bloqueada. Seis animais não tiveram suas falhas preenchidas (Grupo I), seis animais tiveram suas falhas preenchidas com CNQH (Grupo II) e seis animais com CNQH com CTMO (Grupo III), sendo avaliados até 90 dias após a cirurgia. No período pré e pósoperatório (30, 60 e 90 dias) os animais foram submetidos às avaliações radiográficas, sendo as imagens analisadas em relação a efetividade da osteossíntese e a preenchimento da falha por escore, comparando-se os grupos. Após 90 dias, as ovelhas foram submetidas à eutanásia. Observou-se ausência de sinais de toxicidade ou necrose em subcutâneo dos ratos, denotando a biocompatibilidade do compósito e não reação imunogênica às CTMO. A análise histomorfológica por escore da calvária dos ratos demonstrou, em relação ao infiltrado inflamatório que o Grupo Controle apresentou escores mais baixos que os demais grupos, somente aos 20 dias, denotando a diminuição da reação inflamatória ao longo do tempo. O Grupo Controle apresentou pontuações mais baixas de escore aos 20 e 60 dias, para tecido de granulação e neoformação óssea, respectivamente, em relação aos grupos com compósito, que não apresentaram diferenças estatísticas significativas, sugerindo que o compósito auxiliou no processo regenerativo dos defeitos em calvária. Nas avaliações radiográficas em ovinos, os escores de preenchimento de falha nos Grupo I foi menor ao longo do tempo em relação aos Grupos II e III, que apresentaram escores semelhantes. As avaliações histológicas no tecido ósseo de ovinos demonstraram ausência de tecido ósseo neoformado em Grupo I e nos Grupos II e III ausência de reação de corpo estranho, com trabéculas ósseas de tecido neoformado entremeadas ao compósito, em conectividade com tecido pré-existente. Na histomorfometria dos Grupos II e III, observou-se porcentagens semelhantes em relação de tecido ósseo neoformado. Foi possível concluir que o CNQH apresenta biocompatibilidade e perfil osteocondutor para ser utilizado como coadjuvante em casos de perda tecidual óssea.

Research on bone substitutes and bone healing enhancers for critical defects has expanded considerably in recent years. Pre-clinical evaluation of biocompatibility and the osteoconductive potential using in vivo animal models is paramount. Sheep and rats are considered standard models in tissue engineering. Enhancement of bone regeneration can be achieved by using mesenchymal stem cells. The purpose of this study was to evaluate the biocompatibility and osteoconductivity of carbon nanotube/chitosan/hydroxyapatite (CNT/CS/HA) composite scaffolds enriched or not with ovine bone marrow derived mesenchymal stem cells (MSCs) in murine and ovine models. Initially, sixty rats were divided into 2 groups. The CNT/CS/HA composites with or without (MSCs) were implanted in the subcutaneous tissue of 20 animals (euthanized 7 and 30 days after surgical implantation). They were compared to 10 control animals. The CNT/CS/HA composites with or without (MSCs) were also implanted in the calvarial bone defects of another 20 rats (euthanized 20 and 60 days after surgical implantation). They were compared to 10 control animals. Post-mortem histology examinations were performed to compare results between subcutaneous and calvarial tissue groups. At a second moment, we performed tibial ostectomy on eighteen sheep followed by osteosynthesis using locked compression plate. The bone void was left empty on six animals (Group I), filled with CNT/CS/HA on six animals (Group II), and filled with CNT/CS/HA enhanced with MSCs on six animals (Group III). Assessment was performed up to 90 days after surgery. Radiographic assessments were performed before surgery and on days 30, 60 and 90 after surgery. Effectiveness of osteosynthesis and void filling score were compared between groups. The animals were euthanized 90 days after surgery. On post-mortem evaluation the rats subcutaneous tissues were free of any toxicity signs or necrosis. The absence of immunogenic reactions to MSCs denotes the composites biocompatibility. Histomorphological analysis of the rat calvarial score (considering inflammatory infiltrate) showed that Control Group presented lower scores only at the assessment performed on the 20th day after surgery. This finding denotes a decrease trend on the inflammatory reaction over time. Animals from Control Group had lower granulation tissue and bone neoformation scores at days 20 and 60, respectively, when compared to composite groups. Lack of statistically significant differences suggest that the composite material assisted the regeneration of the calvarial defects. On radiographic assessment of the sheep, the void filling scores of Group I were lower than Groups II and III over time. Groups II and III had similar scores. Histological evaluations of the sheeps bone tissues denoted absence of bone neoformation in Group I. Foreign body reaction was not encountered on Groups II and III. Newly formed bone trabeculae entangled to composite and connected to pre-existing tissue were observed. Histomorphometry analysis showed similar newly formed bone tissue ratios in Groups II and III. We concluded the CNT/CS/HA composite is biocompatible and has osteoconductive profile that enables its use as bone enhancer in cases of critical sized bone defects causes by bone tissue loss.